Phytochelatins are small enzyme-synthesized peptides that mediate tolerance to several heavy metals. To gain insight into the unusual metal catalytic site of the Euglena gracilis phytochelatin synthase (EgPCS), site-directed mutagenesis was performed on the C-terminal Cys/His (C/H) rich region 421-C-C-X-H-X-H-X-H-H-H-430. EgPCS mutants were analyzed at the cellular, kinetic and structural levels. Cd2+-resistance was conferred to Cd2+-sensitive yeast cells by EgPCS_wt (476aa) and truncated EgPCS_435 (435aa), but not by EgPCS_415 (415aa), suggesting that the resistance phenotype was linked to the enzyme C/H rich region. Moreover, full-length mutants (in which C and H residues in the C/H rich region were replaced by Ala) EgPCS_C2 (C421A/C422A) and EgPCS_C2H5 (C421A/C422A/H424A/H426A/H428A/H429A/H430A) did not provide Cd2+-resistance; in contrast, EgPCS_H2 (H424A/H426A), EgPCS_H3 (H428A/H429A/H430A), and EgPCS_H5 (H424A/H426A/H428A/H429A/H430A) mutants conferred similar Cd2+-tolerance like EgPCS_wt. Kinetic analysis showed that maximal rate (Vmax) for PC2 synthesis, affinity constants (Km Zn-GS2 or Km Cd-GS2) and catalytic efficiencies (Vmax/Km) were differentially impaired in the mutants, as compared to EgPCS_wt, with EgPCS_C2 being the most perturbed enzyme; however, the K0.5 values for GSH were not affected. All EgPCS mutants were predominantly monomeric. Far UV circular dichroism spectra and differential scanning calorimetry endotherms, indicated that alterations of the catalytic properties of EgPCS_C2 were not due to partial unfolding or destabilization of the native state of this mutant. The results indicated that C421/C422 and H424A/H426A/H428/H429/H430 are essential components of EgPCS for catalysis and activation by metal-substrate complexes.